CM & NH Journal of Cellular Medicine and Natural Health

Cellular Medicine & Natural Health

JOURNAL M. Chatterjee

Comparison of the ef�icacy

of several nutritional
Correspondence to
supplements on cancer Dr. Aleksandra Niedzwiecki,

and normal cell growth

Dr. Rath Research Institute,
1260 Memorex Drive,
Santa Clara, CA 95050,
M. Chatterjee, Ph.D., V. Ivanov, M.D., Ph.D., USA.
A. Niedzwiecki, Ph.D. and M. Rath, M.D.
Dr. Rath Research Institute, CA, USA Email: author@jcmnh.org

Introduction
The majority of scientific and epidemiological evidence supports the beneficial effects of micronutrient supplementation
on various aspects of health. However, there have also been reports that some dietary supplements and vitamins can
promote the growth of cancer. Given that the popularity and use of nutritional supplements is growing, it is important
to conduct a comprehensive evaluation of the efficacy of commercially available nutritional supplements.

Nutritional products on the market vary. Many contain single nutrients, while others contain combinations of
vitamins, minerals, plant extracts and other nutritional components. The efficacy of such products will differ, but
it is rarely, if at all, explained by manufacturers. Usually the selection of specific ingredients is based on published
research conducted on individual vitamin, mineral or other nutritional components, however the biological outcome
of the combination may depend on the synergistic or antagonistic interactions of its ingredients, the source of its raw
materials, and the dose. Manufacturers of nutritional supplements typically do not invest in scientific micronutrient
research, mostly taking care of the technological aspect of the formulations.

It is generally accepted that the quality of ingredients (raw materials) used in nutritional supplements is important in
assuring the efficacy and safety of the products, as many synthetic ingredients have been shown to be harmful or not
effective (1). While some manufacturers promote the use of natural sources in their product‘s ingredients, they do
not provide any evidence that their product demonstrates greater efficacy at the cellular level. As such, in the majority
of multi-nutrient supplements, the structure/function claims of a product are backed up by general statements taken
from the literature in reference to one or a few ingredients tested individually, or purely by marketing slogans.
In the previous study we showed marked differences in the efficacy of different nutritional supplements marketed in
the EU and USA respectively in protecting cells from oxidative stress. This evaluation pointed out the superiority of
synergy-based nutrient combinations (2).

In this report we are comparing in vitro efficacy of various supplements on the growth of cancer cells: melanoma cells
(A2058), liver cancer cells (HepG2) and normal cells (NHDF). The tested formulas were selected from popular, high-
end nutritional supplements sold on the European markets, and nutritional formulas based on nutrient synergy which
were scientifically developed at our Institute. The study focuses on the importance of efficacy-testing nutritional
supplements, not on the commercial aspects, therefore we anonymised all the products.

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Cellular Medicine & Natural Health
JOURNAL M. Chatterjee

Since these products differed not only in nutrient
composition, but also in nutrient doses, we compared
their efficacy based on the recommended daily intake
by the manufacturer of each product, assuming that this
reflects the effective dose of a formulation. Products
were further divided into two subsets, each based on
whether the manufacturers claimed that the ingredients
were based on synergy or the nutrient combination was
random (or not specified by the manufacturer).
See Table 1.

Table 1: Basic composition of tested nutritional supplement formulas

Product Contains Contains Contains Contains Contains Active Synergy

Vitamins Minerals Amino Bioflavonoids/ Compounds claimed
Acids Plant Extracts

SetA #1 Yes (3) Yes (10) Yes (5) Yes (2) Yes (2) Yes

SetA #2 No No No Yes No Yes

SetA #3 Yes (1) Yes (5) Yes (4) Yes (2) No Yes

SetA #4 No No No Yes (7) No Yes

SetB #1 Yes (13) Yes (11) No Yes (1) No No

SetB #2 Yes (13) Yes (11) No Yes (1) No No

SetB #3 Yes (13) Yes (8) No Yes (1) No No

SetB #4 Yes (13) Yes (10) No Yes (2) No No

SetB #5 Yes (13) Yes (7) No Yes (10) Yes (1) No

SetB #6 Yes (12) Yes (9) No Yes (1) Yes (4) No

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Materials and Methods
Cells
Melanoma cells (A2058) were procured from ATCC
(American Type Culture Collection, Rockville, MD, USA)
and maintained in Dulbecco’s Modified Eagle’s Medium
(DMEM) and supplemented with 10% FBS, 100U/ml
penicillin and 100U/ml streptomycin.
HepG2 cells were procured from ATCC (American Type
Culture Collection, Rockville, MD, USA) and maintained
in Dulbecco’s Modified Eagle’s Medium (DMEM) and
supplemented with 10% FBS, 100U/ml penicillin and
100U/ml streptomycin.
Normal human dermal fibroblasts (NHDF) were
supplied by ATCC, maintained in Dulbecco’s Modified
Eagle’s Medium (DMEM) and supplemented with 5%
FBS, 100U/ml penicillin and 100U/ml streptomycin, and
used in experiments at passages 10th to 12th.
Preparation of supplements for testing
All nutritional supplements were treated identically in
accordance with the protocol recommended by Unites
States Pharmacopeia (3). Three recommended daily
doses of each supplement were powdered (tablets were
crushed using ceramic pestle and mortar; capsules were
cut open and powder poured out), placed into a glass
container with 900 ml of 0.1N hydrochloric acid and
incubated for one hour at 37°C in a shaking incubator
set with rotation speed of 75 rpm. Resulting solutions
were filter-sterilized using 0.2 micrometer pore size
filters, aliquoted and kept frozen at -20°C until analyses.
Amounts of samples taken for analysis are expressed as
number of millionth parts of recommended daily dose
of the respective supplement.
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M. Chatterjee
Cell viability assay
The assay was performed using the CellTiter 96®
AQueous One Solution Cell Proliferation Assay (MTS)
supplied by Promega, Madison, WI, USA. This is a
colorimetric assay for assessing cell metabolic activity.
NAD(P)H-dependent cellular oxidoreductase enzymes
in viable cells reduce the tetrazolium dye MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) to formazan. The concentration of formazan
produced is measured by optical density at 490 nm and
it is directly proportional to the number of live cells in
culture.
Cell morphology
Cells were grown in 48 well plates and subjected to
the same conditions as in the cell viability assays and
subsequently fixed with methanol and stained with
Hematoxylin and Eosin dyes. Cells were observed under
a light microscope and representative photos were
taken.
Statistical evaluation:
Results are presented as an average of data obtained from
triplicate or quadruplicate sets. The set number is specified
in each figure. Error bars in each figure represent standard
deviation.
3.
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JOURNAL M. Chatterjee

Results
The results presented on Fig 1 show that the exposure
of melanoma cells to the supplements formulated on
the principles of Synergy (Set A) caused a significant
reduction of the melanoma cell growth. Supplements
A3 and A4 could inhibit melanoma growth completely,
while supplement A#1 by about 20%. In contrast,
five out of six other commercial products had growth
stimulatory effect. Only B#1 showed a pronounced
inhibitory effect on melanoma cells. On average B Set
formulas stimulated melanoma cell growth by 109%.
As presented in Fig 2, cell growth inhibition by the
synergy-based formulas was accompanied by a decrease
in cell viability. The exposure of melanoma cells to all
Set B formulas did not result in marked changes in cell
morphology and viability.
In order to assess whether growth inhibitory and
stimulatory effects were limited to a specific cancer cell
type we tested the effects of the two representative
synergy-based formulas (A#1 and A#3) and five of Set
B commercial products on growth of liver cancer cells
(HepG2 cells). The results on Fig 3 show that similarly
to melanoma cells, the formulas from A and B sets have
different effects on the growth of liver cancer cells.
Formulas A displayed inhibitory effects on liver cancer
cell growth, while all formulas in set B had growth
stimulatory effects (on average by 50%).
Subsequently, we compared the effects of the tested
formulas on the growth of normal cells. The results on
Fig 4 show that formulas in Set A had growth stimulatory
effects ranging from 10-50% on normal human skin cells
(NHDF) while the majority of formulas in Set B inhibited

300
Cell growth as % of Control

250

200

150

100

50

Zero line =Control

control 0

-50

-100
A#1 A#2 A#3 A#4 B#1 B#2 B#3 B#4 B#5 B#6 Average
-150
A#1 A#2 A#3 A#4 B#1 B#2 B#3 B#4 B#5 B#6 Average
Fig 1. Effects of different nutritional supplements on growth of melanoma cells A2058

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Set A

A#1 A#2 A#3

Set B

B#1 B#2 B#4 B#5 B#6

Fig 2. Changes in morphology of melanoma cells A2058 exposed to different nutritional supplement formulas

80

60

40
Cell growth as % of Control

20

Zero line = control 0

No Additions Average

-20

-40

A#2 A#3 B#1 B#2 B#3 B#4 B#5

-60

Fig 3. Effects of different nutritional supplements on growth of hepatocarcinoma cells HepG2

60
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40
 -40

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Cellular Medicine & Natural Health
JOURNAL M. Chatterjee

60

40
Cell growth as % of Control

20

Zero line = control 0

-20
Average
-40

-60
A#1 A#2 A#4 B#1 B#2 B#3 B#4 B#5 B#6

Fig 4. Effects of different nutritional supplements on growth of normal human dermal fibroblast cells

cell growth except for formula B#4. On average B Set
formulas inhibited this normal cell growth by about
20%.
Discussion
With the growing popularity of nutritional supplements
it is vital to evaluate the safety and efficacy of
commercially available products, starting at the cellular
level.
The use of supplements varies in different regions of
the world. It is especially high in Germany and Denmark
(43% and 59% of the adult population respectively take
nutritional supplements) and in the US the CDC reports
that about 40 percent of the population two months of
age and older is taking at least one daily supplement
with some taking multiple supplements a day (4). In
general, women use supplements more than men do
(5, 6, 7, 8, 9).
In the last few years, there have been controversies
surrounding the anti-cancer efficacy of certain vitamins
and nutritional supplements. Various clinical trials or
epidemiological studies have reported positive effects
of multivitamin intake against cancer (10, 11), no
significant effects (12, 13) or even negative outcomes
in people (14). These studies have differed in regard
to tested multi-nutrient compositions, sources of
ingredients in the formulas, doses, the duration of
intake and studied populations.
Therefore in order to eliminate many of these variables
we studied the efficacy of nutritional supplements
applied directly at the level of cells. The in vitro tests
were conducted on melanoma cells (A2058)— a
common skin cancer— and hepatocellular carcinoma
cells (HepG2). For comparison we also used normal
human dermal fibroblast cells, which represent non-
malignant healthy cells abundant in the body. Since,

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the cellular efficacy of the nutritional combinations
may relate to the effective dosage of the products, we
made every effort to evaluate these widely variable
products by applying the same uniform standard. As
such, all formulas were solubilised according to the
method recommended by the US Pharmacopeia and
they were applied according to the doses stated on the
product label. The study did not assess the sources of
raw ingredients in the formulas.
We attribute the consistent anti-cancer effects of
synergy-based formulations to the fact that these
formulas were subjected to scientific studies and
contain carefully selected sources of raw materials.
This deliberate selection of ingredients and scientific
testing could be linked to their recommended daily
dose exhibiting greater safety and efficacy at the cellular
level.
The results imply that the composition and selection of
ingredients as well as correct dosing is vital for achieving
cellular efficacy and safety of a final formula. Therefore
sellers who don’t perform adequate testing of not only
individual ingredients but also their entire formulations
can contribute to the presence of ineffective products on
the market. This not only prevents people from achieving
the health benefits of nutrient supplementation, but
also in the longer term undermines the credibility
of the nutritional supplement industry as a whole,
making people justifiably refrain from taking nutritional
supplements.
We therefore propose that our testing and deliberate
formulation based on the principles of synergy is
important not only for people who take our products,
but for the overall health industry.
This is a limited study for research purposes and does
not take into account batch to batch variations of any of
the tested products. It is possible that results can vary
with different cell types and conditions.
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Conclusions:
The results of this in vitro study indicate that multivitamins
and antioxidant-containing supplements differ in
affecting cancer and normal cell growth. In general, the
formulations based on synergistic nutrient composition
and which have been scientifically developed can
significantly inhibit the growth of cancer cells, while five
out of six of the commercially available formulas tested
in this study displayed different degrees of cancer cell
growth stimulation. The results stress the importance
of scientifically testing nutritional supplements before
making them available to the general public.
Acknowledgments:
The authors appreciate the assistance of Dr. Svetlana
Ivanova in preparing the supplement solutions tested
in the study as well as her participation in valuable
discussions.
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JOURNAL M. Chatterjee

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